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Technical Briefs |
Departments of
1
Child Health,
2 Pathology and Anatomical Sciences, and
3
Internal Medicine, University of Missouri School of Medicine, 1 Hospital Dr., Columbia, MO 65212
4 Kidney & Hypertension Center, 1210 Hicks Blvd., Fairfield, OH 45014
5 Renal Physicians, Inc., 4700 Springboro Pike, Dayton, OH 45439
6 Department of Statistics/Biostatistics, 223 Math Sciences Bldg., University of Missouri, Columbia, MO 65211
aaddress correspondence to this author at: Department of Child Health, M767, University of Missouri School of Medicine, 1 Hospital Dr., Columbia, MO 65212; fax 573-884-8823, e-mail LittleR{at}health.missouri.edu
Many factors can affect interpretation of glycohemoglobin (GHB/HbA1c) measurements in patients with chronic renal failure (CRF). Several reports have suggested that erythrocyte survival is substantially lowered in most patients with CRF; this would be expected to lower GHB results (1)(2)(3)(4)(5)(6). Several reports have also suggested that GHB methods, especially those based on charge separation (e.g., ion-exchange HPLC), may have interference by carbamylated hemoglobin that would be expected to falsely increase GHB results (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Many of these reports evaluated older assay methods; newer ion-exchange methods may show improved separation of the HbA1c fraction from other hemoglobin adducts (15)(17).
Because renal failure is common in patients with diabetes and GHB is widely used as an index of mean blood glucose in these patients, we examined GHB results in patients with CRF by several different GHB assay methods. We also investigated the impact of shortened erythrocyte lifespan by comparing the GHB results obtained for nondiabetic patients with and without CRF.
Fifty-five patients with CRF (blood urea nitrogen >400 mg/L) were recruited for this study. Twenty-nine were not receiving dialysis and were seen at the University of Missouri Nephrology Clinic; the remaining 26 had end stage renal disease and were receiving hemodialysis at the Dialysis Clinic. Patients with renal failure included those with and without diabetes (n = 28 and 27, respectively). The GHB concentration (reported as percentage of HbA1c or equivalent) ranged from 4.4% to 11.2%. Informed consent was obtained from all participants according to University of Missouri-Columbia Health Sciences Institutional Review Board protocol.
Routine clinical specimens from the University of Missouri Hospital and Clinics sent for analysis of GHB as well as specimens from nondiabetic volunteers served as non-CRF control specimens. Control specimens (n = 55) were selected to approximate the GHB range of the CRF samples, and all were confirmed to have blood urea nitrogen <200 mg/L.
All samples were analyzed for GHB with the following methods: Primus CLC330 HPLC (Primus Corporation), Diamat and Variant HPLC (Bio-Rad Laboratories), 2.2 Plus HPLC (Tosoh Medics), and Unimate/Cobas Mira (Roche Diagnostics). A subset of each group of specimens (n = 38) was also analyzed using Variant II HPLC (Bio-Rad Laboratories). The Primus CLC330 was used as the comparative method because carbamylated hemoglobin has been shown not to interfere with affinity chromatography methods (12)(13)(14). Our data from in vitro carbamylated specimens supported this finding; whole blood samples incubated in 5 mmol/L sodium cyanate for 2 h did not show any increase in GHB with the Primus method (R. R. Little, A. L. Tennill, C. Rohlfing, H. M. Wiedmeyer, D. E. Goldstein, unpublished data).
Linear regression analyses were performed between each test method and the comparative method (Primus) for each group of specimens (with or without CRF). An overall test of coincidence was used to determine the statistical significance of CRF on the relationship between each test method and the comparative method, i.e., whether the two regression lines (CRF, test vs comparative method; non-CRF, test vs comparative method) were significantly different. In addition, the slope and intercept for each line were examined separately.
Linear regression analyses were also used to determine whether the presence of carbamylated hemoglobin produced clinically significant interference. Given the Diabetes Control and Complications Trial Reference Method upper limit of normal of 
6% and the American Diabetes Association goal and action limits of 7% and 8%, respectively, GHB (HbA1c) concentrations of 6% and 9% were chosen as important evaluation limits. Given the need to clearly distinguish the difference among 6%, 7%, and 8% GHB, a clinically significant difference was defined as 
0.5% GHB. Both a t-test and a Mann–Whitney test were used to compare GHB results for nondiabetic individuals with and without CRF (measured using the comparative method) to assess whether GHB results for CRF patients were significantly affected by altered erythrocyte lifespan.
Although there was a statistically significant difference (P <0.05) between the regression lines (CRF vs non-CRF) for all methods except the Variant II (Tosoh, P = 0.0058; Diamat, P = 0.0026; Unimate, P = 0.0183; Variant, P = 0.0001; Variant II, P = 0.2338), only the Variant HPLC method showed a clinically significant positive bias (0.59% at 6% GHB, 0.88% at 9% GHB) attributable to CRF (Table 1
). Although it is possible that the smaller sample size used for the Variant II analysis could have provided insufficient power to detect a statistical difference, it is unlikely given the small bias at both 6% and 9% GHB. The difference in results between the Variant HPLC and the comparative method was significantly correlated with blood urea nitrogen (n = 110; r = 0.54; P <0.0001).
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For nondiabetic individuals, the mean ± SD was 5.11% ± 0.44% GHB for the group with CRF (n = 27) and 5.06% ± 0.36% GHB for the group without CRF (n = 30). There was no significant difference in the percentage of GHB between the means of the two groups of nondiabetic individuals (P = 0.63, t-test). Because the distribution of data appeared to be somewhat nongaussian, we also performed a Mann–Whitney test, which showed no significant difference between the medians of the two groups (5.00% vs 4.95%; P = 0.79).
Of the four ion-exchange methods evaluated, only the Variant HPLC showed a clinically significant positive bias in CRF. Unfortunately, there is no simple way of determining which samples have high concentrations of carbamylated hemoglobin during routine measurement of GHB. Each new GHB method should be evaluated for interference from carbamylated hemoglobin; interference cannot be determined based on the method type (e.g., ion-exchange chromatography).
We did not see any evidence of shortened erythrocyte survival in the present group of nondiabetic patients with renal failure. It is possible that, contrary to some reports, most patients with CRF do not have shortened erythrocyte survival. Alternatively, impaired glucose control in CRF may have offset the effect of shortened erythrocyte survival.
We conclude that GHB can provide valid results for most patients with CRF if an appropriate methodology is used.
Acknowledgments
This study was funded in part by Tosoh Medics, Inc. (South San Francisco, CA).
References
The following articles in journals at HighWire Press have cited this article:
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  S. Kazempour-Ardebili, V. L. Lecamwasam, T. Dassanyake, A. H. Frankel, F. W.K. Tam, A. Dornhorst, G. Frost, and J. J.O. Turner Assessing Glycemic Control in Maintenance Hemodialysis Patients With Type 2 Diabetes Diabetes Care, July 1, 2009; 32(7): 1137 - 1142. [Abstract] [Full Text] [PDF]
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 E S Kilpatrick Haemoglobin A1c in the diagnosis and monitoring of diabetes mellitus J. Clin. Pathol., September 1, 2008; 61(9): 977 - 982. [Abstract] [Full Text] [PDF]
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D. J. Tascona, A. R. Morton, E. B. Toffelmire, D. C. Holland, and E. A. Iliescu Adequacy of Glycemic Control in Hemodialysis Patients With Diabetes Diabetes Care, October 1, 2006; 29(10): 2247 - 2251. [Abstract] [Full Text] [PDF]
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V. Rigalleau, C. Lasseur, C. Raffaitin, C. Perlemoine, N. Barthe, P. Chauveau, C. Combe, and H. Gin Glucose Control Influences Glomerular Filtration Rate and Its Prediction in Diabetic Subjects Diabetes Care, July 1, 2006; 29(7): 1491 - 1495. [Abstract] [Full Text] [PDF]
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 W. Hoelzel, C. Weykamp, J.-O. Jeppsson, K. Miedema, J. R. Barr, I. Goodall, T. Hoshino, W. G. John, U. Kobold, R. Little, et al. IFCC Reference System for Measurement of Hemoglobin A1c in Human Blood and the National Standardization Schemes in the United States, Japan, and Sweden: A Method-Comparison Study Clin. Chem., January 1, 2004; 50(1): 166 - 174. [Abstract] [Full Text] [PDF]
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